β-Lactamase Tools for Establishing Cell Internalization and Cytosolic Delivery of Cell Penetrating Peptides

Biomolecules
Shane R StoneNadia Milech

Abstract

The ability of cell penetrating peptides (CPPs) to deliver biologically relevant cargos into cells is becoming more important as targets in the intracellular space continue to be explored. We have developed two assays based on CPP-dependent, intracellular delivery of TEM-1 β-lactamase enzyme, a functional biological molecule comparable in size to many protein therapeutics. The first assay focuses on the delivery of full-length β-lactamase to evaluate the internalization potential of a CPP sequence. The second assay uses a split-protein system where one component of β-lactamase is constitutively expressed in the cytoplasm of a stable cell line and the other component is delivered by a CPP. The delivery of a split β-lactamase component evaluates the cytosolic delivery capacity of a CPP. We demonstrate that these assays are rapid, flexible and have potential for use with any cell type and CPP sequence. Both assays are validated using canonical and novel CPPs, with limits of detection from <500 nM to 1 &micro;M. Together, the β-lactamase assays provide compatible tools for functional characterization of CPP activity and the delivery of biological cargos into cells.

References

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Citations

Feb 10, 2021·Advanced Drug Delivery Reviews·Macarena Sánchez-Navarro
May 16, 2019·Chemical Reviews·Patrick G DoughertyDehua Pei

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Methods Mentioned

BETA
fluorescence resonance
fluorescence microscopy
confocal microscopy
transfection
imaging techniques
FCS
flow cytometry
size exclusion chromatography
Protein Assay
electrophoresis

Software Mentioned

Phylomer
FlowJo X
FACSDiva
Prism
GraphPad

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